Reversible protein phosphorylation is one of the most pervasive strategies for regulation in biological systems. An example of this form of regulation is provided by the regulatory phosphorylation of isocitrate dehydrogenase (IDH) of Escherichia coli. This has been chosen as the experimental model for this study primarily because of the potential for the use of molecular genetics and recombinant DNA technology in probing the system. The ultimate goal of this project is to achieve an understanding of the functioning of the phosphorylation system in the intact organism. This will be accomplished by characterizing the behavior of the individual components and using this knowledge to analyze the functioning of the system in vivo. Studies performed under this proposal will concern the structure/function relationships of IDH kinase and phosphatase and the bahavior of these activities in vivo. IDH kinase and phosphatase have been found to be physically associated, probably on the same polypeptide chain. In an effort to determine the regulatory and structural significance of this bifunctionality, the relationships between the functional domains will be characterized. In addition to the methods of protein chemistry, this study will make extensive use of recombinant DNA technology, including in vitro mutagenesis. Specific problems to be addressed include the evolutionary relationship between the domains, the degree to which they are physically autonomous and the potential for inter-domain allosteric communication. The clones of the IDH kinase/phosphatase gene and of its genetically altered derivatives which will be developed and characterized in the structural studies, will provide the basis for study of the system in vivo. In this project, variation in the cellular level of IDH kinase/phosphatase will be used to define the factors which are responsible for the rate at which the phosphorylation system responds to stimulation.